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FEBS Letters 581 (2007) 4910–4916

Two distinct human POM121 : Requirement for the formation of complexes

Tomoko Funakoshia, Kazuhiro Maeshimaa, Kazuhide Yahatab, Sumio Suganoc, Fumio Imamotob, Naoko Imamotoa,* a Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan b Department of Molecular Biology, BIKEN, Osaka University, Osaka, Japan c The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Received 30 March 2007; revised 12 September 2007; accepted 12 September 2007

Available online 21 September 2007

Edited by Ulrike Kutay

essential in somatic cells, as this is not ubiquitously ex- Abstract Pom121 is one of the integral membrane components of the nuclear pore complex (NPC) in vertebrate cells. Unlike ro- pressed in mammalian cells [6]. Pom121 is recruited at an early dent cells carrying a single POM121 , human cells possess stage of post-mitotic nuclear reassembly in vertebrate cells, multiple POM121 gene loci on 7q11.23, as a conse- and its turnover on the interphase NPC is very slow [7]. These quence of complex segmental-duplications in this region during findings make Pom121 a strong candidate that could possess human evolution. In HeLa cells, two ‘‘full-length’’ Pom121 are essential roles in anchoring the NPC to the NE. Indeed, transcribed and translated by two distinct genetic loci. RNAi Pom121 was shown to be essential for NPC formation, but experiments showed that efficient depletion of both Pom121 pro- not gp210 in the Xenopus egg extracts [8]. The human ortholog teins significantly reduces assembled NPCs on . of yeast NDC1, which was initially isolated as a component of Pom121-depletion also induced clustering of NPCs, indicating spindle pole bodies in yeast, localizes to the NE in HeLa cells its role on maintenance of NPC structure/organization. during interphase [9,10]. Recently, two groups reported the 2007 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. essential roles of Ndc1 for NPC assembly in mammalian cells, but controversial results were reported for Pom121 [8,9,11].It Keywords: Nuclear pore complex; Pom121; Nuclear structure; is still unclear whether Pom121 is necessary for NPC assembly RNAi; Segmental-duplication or not in mammalian somatic cells. In this study, we found that human ‘‘full-length’’ Pom121 are encoded by two distinct genetic loci, both of which are transcribed and translated in HeLa cells. Using several RNAi oligos targeted to both or either of Pom121 transcripts, we 1. Introduction show that depletion of Pom121 causes a reduction of assem- bled NPCs, and clustering of NPCs. Our results show necessity Nuclear pore complex (NPC) mediates the transport of mac- of Pom121 on NPC assembly and maintenance of NPC struc- romolecules across the nuclear envelope (NE) [1]. The NPC is ture/organization. a very large protein complex with an estimated mass of 60– 125 MDa in vertebrates and is assembled from multiple copies of approximately 30 different termed 2. Materials and methods (Nups) [2]. NPCs are dynamic structures that undergo assem- bly and disassembly upon cell cycle progression. NPC assem- 2.1. Isolation of two distinct human POM121 cDNAs and expressions of bly takes place not only at the end of mitosis when the NE their Venus-fusion genes in HeLa cells reforms around daughter , but also during inter- POM121 cDNA was obtained from HeLa S3 total RNA using the 50 phase on the assembled NE [3,4]. RACE system (Invitrogen) according to the manufacture’s instruc- tions. First-strand cDNA was synthesized with SuperScript III re- The integral membrane Nups are believed to play an impor- verse transcriptase (Invitrogen) and POM121 specific primer tant role in NPC formation and anchoring of the other Nups (Supplementary materials). Poly-dA tail was added to the first-strand to the NE. In vertebrates, only three Nups, Pom121, gp210 cDNA and used as a template for second-strand synthesis using an oli- and NDC1 are known as transmembrane proteins. gp210 is go-(dT)17 primer. POM121 cDNAs were amplified by PCR with LA- Taq DNA polymerase (TaKaRa) using primer sets corresponding to implicated in NPC formation, since an antibody against its POM1211–24 and POM121837–855 at locus A, and POM1211–24 and short C-terminal peptide and the peptide itself both inhibited POM1211180–1201 at locus C. The amplified DNA-fragments were ver- NPC formation in the Xenopus egg extract [5]. However, the ified by DNA sequencing and fused with a truncated POM121 cDNA function of gp210 in NPC assembly is considered to be non- clone (GenBank accession nos: BC008794) to obtain cDNA coding the full-length of human Pom121 proteins. For the expression of POM121 cDNAs, pEXPR-PEF-1a-POM121A-Venus, and pEXPR-PEF-a- *Corresponding author. Fax: +81 48 462 4715. POM121NC-Venus were constructed based on multi-site Gateway E-mail address: [email protected] (N. Imamoto). system (Invitrogen) as described previously [3,12]. To express POM121-Venus and CFP simultaneously, tandem expression clones, Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; pEXPR-PEF-1a-POM121A-Venus-Ix2-PEF-1a-SECFP, pEXPR-PEF-1a- Nup, ; RT-PCR, reverse transcriptase-polymerase chain POM121NC-Venus-Ix2-PEF-1a-SECFP were constructed with tandem reaction; siRNA, small interfering RNA insulator sequences (Ix2) that ensure comparable expressions of

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.09.021 T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916 4911

POM121-Venus and SECFP [13]. Ix2 insulator consists of two repeti- the C-terminal portion of rodent Pom121, but its predicted N- tive chicken b-globin HS4 insulators (a gift from Dr. Felsenfeld). Sta- terminal sequence shows no homology with Pom121. This is ble and transient transformants expressing POM121-Venus were due to a truncation of segmental-duplication block at the cen- obtained as previously described [3,12]. tromeric terminal on locus B. A deduced 1229-aa polypeptide 2.2. RNAi transfection encoded by locus C harbors an N-terminal hydrophobic trans- The cells plated at 1 · 104 cells in 24-well plates were transfected membrane domain, but no information of a cDNA encoding with 50 nM small interfering RNAs (siRNAs) with Oligofectamine ‘‘full-length’’ Pom121 protein derived from this locus is avail- reagent (Invitrogen) according to the manufacture’s instructions. After able in the databases with the exception of deduced sequences 72 h, the cells were processed for indirect immunofluorescence. For from the genome. studies in Fig. 3, cells were synchronized by double thymidine treat- ment (2 mM, 16 h) in order to obtain uniform expression levels and nu- clear rim targeting of transfected Pom121. The cells on a 35 mm-dish 3.2. Identification and expression analysis of a new exon were co-transfected with 2 lg plasmid and 48 nM RNA oligo, after encoding a transmembrane domain of POM121 1st thymidine treatments. Twenty-seven hours after 2nd thymidine The lack of the 50-portion of human POM121 cDNA that treatment (44-h after co-transfection), middle section images (0.2 lm) were obtained. Fluorescence intensities of Venus of nuclear rims of encodes the N-terminal region containing the transmembrane the cells expressing SECFP at almost the same level were measured domain prompted us to re-examine the genetic loci. We sur- using SoftWorx software package (Applied Precision). veyed the genomic sequence of locus A that shows homology The sequences of siRNAs used are indicated in Supplementary mate- with the N-terminal region of rodent Pom121. A candidate se- rials. quence was present within a long intron between the original exon 4 and exon 5 (Fig. 1B). The sequence segment encodes 2.3. Immunofluorescence staining Immunofluorescence was carried out as described previously [3] ex- an ORF homologous to the first 189 amino acids of rodent cept that methanol-fixed cells were used in all cases. Antibody dilution Pom121 protein including the transmembrane region (supple- rates used are following: 1:3000 dilution for mAb414 (Covance, MMS- mentary Fig. S1C). Exon/intron boundary consensus se- 120P); 1:1000 dilution for Pom121, lamin A/C (Santa Cruz, sc-20681), quences were found on both sides of this segment and the and goat secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 (Invitrogen). spliced junction sequence followed the GT-AG rule, indicating that this region contains an exon that was dismissed in the pre- vious annotation. We referred this putative exon as exon 4a. A 3. Results and discussion genomic sequence similar to exon 4a also exists in locus C, but not in locus B (supplementary Fig. S1B). Successful amplifica- 3.1. Human POM121 is assigned on three genetic loci on tion of three small regions using reverse transcriptase-polymer- chromosome 7q11.23 ase chain reaction (RT-PCR) from locus A and C, which Pom121 is a well-conserved transmembrane NPC compo- contain small ‘‘gaps’’ missing in one gene from another, indi- nent present in a wide range of vertebrate species, and is cated that ‘‘full-length’’ POM121 from both locus A and C thought to serve as the anchoring sites of the vertebrate are transcribed in HeLa cells (supplementary Fig. S2A, B). NPC to the lipid bilayers. To obtain a putative complete se- In addition, several unique peptide fragments corresponding quence of the human POM121 gene, we searched the Gen- to the both POM121 products were detected by mass spec- Bank/EMBL databases. Unlike rodent cells carrying a single trometry analysis of nuclear pore fraction isolated from HeLa POM121 gene, we found that human cells possess three differ- cells (supplementary Fig. S2C), indicating that Pom121 pro- ent genetic loci on chromosome 7q11.23 within a range of teins are expressed from both locus A and C. 3.3-Mb, designated here as locus A, B and C (Fig. 1A). Each locus potentially encodes a long open reading frame (ORF) 3.3. Cloning and characterization of 50-upstream region of that shows a high homology to rodent POM121. The chromo- human POM121 gene somal environments of human POM121 gene loci resemble to DNA fragments containing exon 4a and a portion of exon 5 those of rat and mouse POM121 gene loci, although numerous of locus A, as well as the corresponding region of locus C were duplications and inversions are seen in the case of human gene amplified from HeLa cDNA library, cloned, and sequenced (supplementary Fig. S1A). Human chromosome 7q11.23 is (GenBank accession nos: AB289621, AB354586, Fig. 1C). implicated in Williams–Beuren syndrome, a segmental aneu- Exon 4a of locus A consists of 644-nucleotides showing 95% somy syndrome caused by the frequent deletions of DNA and 74% identities to the corresponding regions of locus C segments. This chromosome region was reported to have and the reported sequence of rat POM121, respectively. large-scale chromosomal rearrangements accompanying evolu- To characterize the cDNA fragments cloned in this study, tionary inversions of the genome and the duplications of large we expressed in HeLa cells a full-length Pom121 tagged with (300 kb) blocks of DNA before the diversification of homi- a bright YFP-derivative, Venus. Both proteins containing N- noids [14]. Each of three POM121 loci locates within one of terminus of locus A and C clearly localized to the nuclear such segmental duplicated blocks (Fig. 1A, black arrows). rim, and most of the punctuate dot signals were co-localized In spite of having multiple POM121 gene loci in the human with mAb414 antibody staining signals that represent NPCs, genome, there exist no cDNA clones coding ‘‘full-length’’ hu- as well as endogenous Pom121 signals detected by antibodies man Pom121 registered in current public databases (GenBank/ prepared in this study (Fig. 2). On the other hand, a Venus-fu- EMBL, see supplementary Fig. S1). For example, all clones as- sion protein from the previously registered cDNA clones, mi- signed to human POM121 cDNA lack N-terminal transmem- grated into the nuclei, but failed to target the nuclear rim brane region, a portion reported to be important for function (supplementary Fig. S3). These results show that N-terminal of rodent POM121 as integral membrane protein [15]. At locus portion identified in this study is indeed necessary for B, an ORF encodes an 856-aa showing a high homology with Pom121 to target NPCs. 4912 T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916

Fig. 1. Human POM121 genes. (A) Schematic representation of POM121 genes on human chromosome 7q11.23. The relative positions of several known genes (white arrows), and relative locations of the centromere (CEN) and telomere (TEL) are indicated. The segmental-duplications blocks carrying the POM121 locus A, B and C are boxed. (B) Schematic representation of the exon organizations of human POM121 gene at locus A. Boxes and horizontal lines indicate exons and introns of POM121 gene organization, respectively. The untranslational and coding regions are indicated as open and filled boxes, respectively. The new coding exon identified in this study, exon 4a, is indicated as a shaded box. Alternative splicing variants from locus A contain either exon 16 or 16a (gray) (see Supplementary Fig. S1B). The first methionines of the full-length and N-terminally truncated products are shown as M1 and M2, respectively. Corresponding to that regions of cDNAs cloned in this study are underlined. (C) Nucleotide sequences of cDNA cloned in this study. upper: Pom121A, lower; Pom121C. The targeting sequence of RNAi oligo-1, showing two nucleotides miss match, used in Figs. 3 and 4 is underlined.

We did not detect the expression of N-terminally truncated 3.4. Knockdown of Pom121 causes the disappearance of NPC on Pom121 by immunoblotting in HeLa cells (Fig. 2C). Since the NE and aberrant nuclear morphologies the truncated POM121 cDNA clones registered in the dat- To examine the cellular function of Pom121s, small interfer- abases contain a consensus Kozak sequence upstream of their ing RNAs (siRNAs) for Pom121 were transfected into HeLa initiation codon, possibility that truncated Pom121 protein cells to deplete endogenous Pom121. For this, we initially de- might be expressed in some other human cells/tissues cannot signed 4 different oligo RNA duplexes: two of which show be excluded. complete sequence match only with the transcript derived from T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916 4913

Fig. 2. Localization and expression of Pom121s. Rat anti-Pom121 serum was obtained after immunization of bacterially expressed Pom121A 433- 754 aa, and used for cell staining. (A) Distribution of endogenous Pom121. Nuclear surface images of HeLa cells co-stained with anti-Pom121 serum (a) and mAb414 antibody staining NPC (b) are shown. Merged images (column c): green, Pom121; red, mAb414. Bar shows 10 lm. (B and C) Nuclear surface images of the cells expressing either Pom121A (full-length)-Venus (a in row B) or Pom121-Venus with N-terminus (1–398 aa) of Pom121C (Pom121NC) (a in row C). The staining by the mAb414 antibody (NPC) is shown in column b. Magnified images are shown in the insets. (D) Western blotting of whole HeLa cell lysate (5 · 104 cells/lane). Lane 1, pre-immune serum (PI); Lane 2, anti-Pom121 serum pre-incubated with only buffer; Lane 3, serum with 0.001 mg/ml recombinant Pom121A (433–754 aa) peptide; Lane 4, serum with 0.01 mg/ml peptide; Lane 5, serum with 0.1 mg/ml peptide; Lane 6 serum with 0.1 mg/ml BSA (control). Note that a signal detected at a position of about 130 kDa is non-specific because it did not disappear in the presence of excess antigen (shown as asterisk).

either locus A or locus C (Fig. 3 oligo-1A and -1C, see also efficient with oligo-2. On the other hand, oligo-1A and -1C Fig. 1C) and the other two oligos have complete match to both preferentially depleted POM121A and POM121C, respectively transcripts from locus A and C (Fig. 3 oligo-2 and -3). To (Fig. 3B). examine their knockdown specificity and efficiency, each oligo We next examined cellular effects of endogenous Pom121- was transfected to the HeLa cells transiently co-expressing depletion using above oligos in HeLa cells. As shown in either Pom121A-Venus and CFP (expression marker), or Fig. 4 (4th and 5th rows), treatment with oligo-2 and oligo- Pom121NC-Venus (Pom121 possessing N-terminus sequence 3, which target both Pom121A and C, effectively depleted of Pom121C) and CFP. As shown in Fig. 3, oligo-2 and -3 suc- endogenous Pom121 proteins to an almost undetectable level cessfully reduced the expression levels of both Pom121A-Ve- by immunofluorescence. This level of Pom121-depletion nus and Pom121NC-Venus, although the depletion was more caused a decrease in the mAb414 staining signals of NPCs 4914 T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916

Fig. 3. Verifications of targeting specificity and depletion efficiency of various RNA oligos used in Pom121-knockdown experiments. (A) Target positions of RNAi oligo used in this study. Oligo-1A and -1C show complete match with Pom121A and Pom121C, respectively, but contain two- nucleotides miss match with Pom121C and Pom121A, respectively. Oligo-2 and -3 show complete match with both Pom121A and C. (B) Efficiency of Pom121-depletion. The HeLa cells co-transfected with tandem expression clone of Pom121A-Venus/CFP (upper) or Pom121NC-Venus/CFP (lower) with indicated RNA duplexes during double thymidine block treatment. 44 h after transfection of each oligo, fluorescence intensity of Venus was examined in the cells expressing CFP. Panels show live-images of the cells. All the images were taken with same exposure time. Note that oligo-1A and -1C preferentially depleted Pom121A and Pom121NC, respectively, whereas oligo-2 and -3 removed both Pom121A and Pom121NC with almost same efficiency. (C) Quantificative analysis of panel B. The integrated fluorescence intensities in certain areas of the 10–20 nuclei expressing Pom121A-Venus/CFP or Pom121NC-Venus/CFP transfected with the various RNA oligos were measured. Relative fluorescent ratios (RNA oligo- transfected cells/ control cells) are plotted. Values are expressed as means ± S.D.

(see also supplementary Fig. S4 for comparison with control these two oligos were below the detection level when judged cells). Although depletion level of endogenous Pom121 with by immuno-staining signals, the differences in the depletion le- T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916 4915

Fig. 4. Cellular effects of Pom121-depletion by RNAi. HeLa cells transfected with either Pom121 oligo-1A, oligo-1C, oligo-2, or oligo-3 RNA duplex were fixed, and immunostained with the anti-Pom121 rat serum (Pom121), mAb414 antibody (NPC), and anti-lamin A/C antibody (Lamin A/C). As controls, cells were treated with only the transfection regent (Control), or with RNA duplexes targeted to hsc70 (hsc70). Nuclear structures are represented by DAPI staining (DNA). Bar, 10 lm. Magnified images (·4) of mAb414 staining are shown in the insets. Values in Pom121 and NPC panels show depletion levels (% of control) examined by fluorescent intensity of Pom121 and mAb414 staining signals measured. In all the cases, mean fluorescent intensity of Pom121 signals was obtained from the images of over 10 nuclei that were taken with the same exposure time.

vel of mAb414 signals among oligo-2 and oligo-3 could be a or oligo-1C (3rd row), which targets preferentially one of consequent of their efficiency to knock-down Pom121 as Pom121 proteins, reduced the endogenous Pom121 level, but shown in Fig. 3 (quantification results show slightly lower with lower efficiency compared to oligo-2 and -3 (judged by knock-down efficiency for oligo-3, when compared to oligo- immuno-staining signals, see Fig. 4). In this case, NPCs did 2). On the other hands, treatment with oligo-1A (2nd row) not disappear, but their clustering on the NE was prominent 4916 T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916

(Fig. 4). It is notable that NPCs still remaining with the trans- References fection of oligo-2 and -3 also tend to cluster (see magnified views in Fig. 4). We could not establish specific antibodies that [1] Imamoto, N. (2000) Diversity in nucleocytoplasmic transport recognize only Pom121A or C, because of their high sequence pathways. Cell Struct. Funct. 25, 207–216. [2] Suntharalingam, M. and Wente, S.R. (2003) Peering through the homologies throughout the entire protein (96% identical, sup- pore: nuclear pore complex structure, assembly, and function. plementary Fig. S2C). However, since oligo-1A and -1C de- Dev. Cell 4, 775–789. pleted one of Pom121 products efficiently (Fig. 3), it is very [3] Maeshima, K., Yahata, K., Sasaki, Y., Nakatomi, R., Tachibana, likely that Pom121A and Pom121C remained respectively in T., Hashikawa, T., Imamoto, F. and Imamoto, N. (2006) Cell- cycle-dependent dynamics of nuclear pores: pore-free islands and oligo-1C and -1A-transfected HeLa cells. Reduction of NPC lamins. J. Cell Sci. 119, 4442–4451. signals, as well as NPC clustering, was specific to Pom121- [4] D’Angelo, M.A., Anderson, D.J., Richard, E. and Hetzer, M.W. depletion, not due to the secondary effects of growth arrest (2006) Nuclear pores form de novo from both sides of the nuclear or cell death, because depletion of hsc70 [16], which also leads envelope. Science 312, 440–443. to cell death did not show such effects on NPCs. [5] Drummond, S.P. and Wilson, K.L. (2002) Interference with the cytoplasmic tail of gp210 disrupts ‘‘close apposition’’ of nuclear Another obvious effect observed upon Pom121-depletion membranes and blocks nuclear pore dilation. J. Cell Biol. 158, 53– was the induction of abnormally shaped nuclei. The shapes 62. of nuclei in the cells used in the present study are round and [6] Olsson, M., Scheele, S. and Ekblom, P. (2004) Limited expression uniform, as evidenced by DAPI staining of control cells. With of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans. Exp. Pom121-depletion, the nuclear shapes became irregular Cell Res. 292, 359–370. (Fig. 4) in all of the cells examined. Nuclear lamins are known [7] Rabut, G., Doye, V. and Ellenberg, J. (2004) Mapping the to be important in maintaining nuclear morphology. Treat- dynamic organization of the nuclear pore complex inside single ment with the Pom121 siRNA duplex had no effect on the pro- living cells. Nat. Cell Biol. 6, 1114–1121. tein levels of lamins (supplementary Fig. S5). Apparent [8] Antonin, W., Franz, C., Haselmann, U., Antony, C. and Mattaj, I.W. (2005) The integral membrane nucleoporin pom121 func- clustering of lamin A/C on NE observed upon Pom121-deple- tionally links nuclear pore complex assembly and nuclear enve- tion is likely to be a consequent of irregular nuclear morphol- lope formation. Mol. Cell 17, 83–92. ogy (see supplementary Movie 1). [9] Mansfeld, J., Guttinger, S., Hawryluk-Gara, L.A., Pante, N., Recently, two groups reported controversial results on the Mall, M., Galy, V., Haselmann, U., Muhlhausser, P., Wozniak, R.W., Mattaj, I.W., Kutay, U. and Antonin, W. (2006) The necessity of mammalian Pom121 for NPC assembly on the conserved transmembrane nucleoporin NDC1 is required for NE using RNAi [8,9,11]. In present study, we found there exist nuclear pore complex assembly in vertebrate cells. Mol. Cell 22, two Pom121 proteins expressing from different genetic loci in 93–103. human cells, and showed that NPC reduction strictly depends [10] Stavru, F., Hulsmann, B.B., Spang, A., Hartmann, E., Cordes, on the amount of both Pom121 proteins remaining in the cells. V.C. and Gorlich, D. (2006) NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes. J. Cell Biol. Depletion of Pom121 also induces NPC clustering, which 173, 509–519. implicate a role of this protein in maintenance of NPC struc- [11] Stavru, F., Nautrup-Pedersen, G., Cordes, V.C. and Gorlich, D. ture/organization that need to be clarified in the future study. (2006) Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells. J. Cell Biol. 173, 477–483. Acknowledgements: We are grateful to Ms Ichikawa and Ms Nakaza- [12] Yahata, K., Kishine, H., Sone, T., Sasaki, Y., Hotta, J., Chesnut, wa for DNA sequencing (Bioarchitect Research Group, RIKEN), Ms J.D., Okabe, M. and Imamoto, F. (2005) Multi-gene gateway Kaori Otsuki and Mr Masaya Usui (BSI’s Research Resources Center, clone design for expression of multiple heterologous genes in RIKEN) for mass spectrometry analysis, and Ms Shimazaki for tech- living cells: conditional at near physiological nical assistance. We also thank Drs Miyawaki and Felsenfeld for their levels. J. Biotechnol. 118, 123–134. generous gifts of Venus cDNA and chicken HS4 sequence, respec- [13] Yahata, K., Maeshima, K., Sone, T., Ando, T., Okabe, M., tively. This work was supported by Grants-in-Aid from the Ministry Imamoto, N. and Imamoto, F. (2007) cHS4 insulator-mediated of Education, Science, Sports and Culture of Japan and partly by a alleviation of promoter interference during cell based expression fund from the Bioarchitect Research Project, Chemical Biology Pro- of tandemly associated transgenes. J. Mol. Biol., in press. ject, and Strategic Program for R&D of RIKEN. [14] Antonell, A., de Luis, O., Domingo-Roura, X. and Perez-Jurado, L.A. (2005) Evolutionary mechanisms shaping the genomic structure of the Williams-Beuren syndrome chromosomal region at human 7q11.23. Genome Res. 15, 1179–1188. [15] Imreh, G., Maksel, D., de Monvel, J.B., Branden, L. and Appendix A. Supplementary data Hallberg, E. 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